title: Random PCR and ultracentrifugation increases sensitivity and throughput of VIDISCA for screening of pathogens in clinical specimens.
abstract: BACKGROUND
  Virus discovery based on cDNA-AFLP (VIDISCA) is a sequence-independent virus discovery method that was recently developed and successfully used to characterize unknown viruses in cell cultures. Its applicability, however, is limited by its low sensitivity.
  
  
  METHODS
  We evaluated whether the introduction of prior amplification of target sequences by random PCR (rPCR) increases the sensitivity of this method to improve its use on clinical specimens. In addition, ultracentrifugation was added to the protocol to allow for pooling of multiple samples, thereby increasing analytical throughput of the VIDSCA.
  
  
  RESULTS
  We showed that rPCR enhanced the sensitivity of VIDISCA by 100-fold for two out of four viruses in different clinical samples, and that the ultracentrifugation step allowed for analyzing samples of large volumes (4 ml) and simultaneous processing of multiple (~40) clinical specimens.
  
  
  CONCLUSIONS
  We conclude that this modified VIDISCA protocol is a relatively easy method to use for screening of large numbers of clinical samples that are suspected to contain previously unrecognized pathogens, in settings where ultradeep sequencing platforms are not available.
authors.affiliation: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.tanlv@oucru.org
authors.forename: Le Van
authors.initials: le V
authors.lastname: Tan
authors.forename: H Rogier
authors.initials: HR
authors.lastname: Van Doorn
authors.forename: Lia
authors.initials: L
authors.lastname: Van der Hoek
authors.forename: Vo
authors.initials: V
authors.lastname: Minh Hien
authors.forename: Maarten F
authors.initials: MF
authors.lastname: Jebbink
authors.forename: Do
authors.initials: D
authors.lastname: Quang Ha
authors.forename: Jeremy
authors.initials: J
authors.lastname: Farrar
authors.forename: Nguyen
authors.initials: N
authors.lastname: Van Vinh Chau
authors.forename: Menno D
authors.initials: MD
authors.lastname: de Jong
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D008403:Mass Screening / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014461:Ultracentrifugation; D014773:Virology / Q000379:methods; D014777:Virus Diseases / Q000175:diagnosis* / Q000821:virology*; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; C055927:endodeoxyribonuclease MseI; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases